Maintenance of normal hemostasis requires a delicate balance of the pro- and anti-coagulant mechanisms that are involved in blood coagulation. A dysfunction of one of the proteins may result in bleeding tendencies or thrombotic events. A molecular defect in one of the pro-coagulant proteins is commonly associated with bleeding tendencies, that can be overcome by replacement therapy. This is best illustrated by the bleeding disorder haemophilia A, which is associated with a functional absence of Factor VIII, an essential cofactor in the conversion of Factor X to factor Xa, by activated Factor IX (Kane and Davie. 1988. Blood, vol. 71, 539-555). Diagnosis of bleeding tendencies is performed by simple laboratory tests which are well known in the art. In addition, more specific assays have been developed employing chromogenic substrates in conjunction with purified coagulation Factors that are used to monitor the precise levels of several pro-coagulant proteins. Currently, adequate diagnostic techniques are available to monitor the majority of deficiencies observed in patients with bleeding tendencies.
The anticoagulant pathway, ultimately resulting in the inactivation of the pro-coagulant cofactors V and VIII, by APC, has been described in considerable detail (Esmon, C. T. 1993, Thromb. Haemost. vol. 70, 29-35). Protein S has been implicated as a cofactor in the inactivation of both Factor V and VIII, although the effect of protein S on the catalytic efficiency of cleavage of both Factor V and VIII is relatively small (Koedam et al., 1988, J. Clin. Invest. vol. 82, 1236-1243; Kalafatis and Mann. 1993. J. Biol. Chem., vol. 268, 27246-27257). Functional absence of one of the proteins involved in the anticoagulant pathway is commonly associated with thrombosis. Molecular defects in several proteins involved in the anti-coagulant pathway have found to be associated with thrombotic events. Homozygous protein C deficiency clearly is associated with severe thrombotic events which can be corrected by replacement-therapy (Dreyfus et al., 1991, N. Eng. J. Med. vol. 325, 1565-1568). Heterozygous protein C deficiency has also been established as an increased risk for thrombosis (Bertina et al., 1982, Thromb. Haemost. vol. 48, 1-5), although additional factors seem to be involved in at least some cases (Miletich et al. 1987, N. Eng. J. Med. vol. 317, 991-996). Similar to protein C, protein S deficiency is associated with an increased risk of thrombosis (Comp et al., 1980, J. Clin. Invest. vol. 74, 2082-2088). Relatively rare genetic defects in anti-thrombin III, fibrinogen and plasminogen have also been implicated in thrombosis. Taken together, several deficiencies of proteins involved in the anti-coagulant pathway have been associated with an increased risk of thrombosis. However, the deficiencies outlined above offer an explanation in no more than 10 to 30% of patients suffering from thrombo-embolic disease, while the remainder of the cases remains unexplained (Heijboer et al., 1990, N. Eng. J. Med. 22, 1512-1516). Thus, diagnosis of patients suffering from thrombo-embolic disease is inadequate in 70 to 90% of the cases. Recent advances have decreased the percentage of unexplained thrombosis to 40 to 60%. Dahlback and co-workers have observed resistance to APC in a patient suffering from multiple thrombotic events (Dahlback et al., 1993, Proc. Natl. Acad. Sci. USA, vol. 90, 1004-1008). An assay based upon the prolongation of the clotting-time by APC, as measured in the activated partial thromboplastin time (APTT) was used to analyze the defect. No prolongation of the APTT upon addition of APC was observed, indicating a defect in the anti-coagulant pathway. Other groups have confirmed the occurrence of APC-resistance in patients suffering from deep vein thrombosis and larger studies performed indicate that 20 to 40% of patients suffering from thrombotic episodes, display resistance to APC (Griffin et al., 1993, Blood, vol. 82, 1989-1993; Koster et al., 1993, Lancet vol., 342, 1503-1506).
The phenotype of APC-resistance is not limited to patients suffering from venous thrombosis. Several studies have documented that the prevalence of APC-resistance is about 2-5% in the normal population. The molecular basis of APC-resistance has remained obscure for some time. A recent study revealed that the phenotype of APC-resistance could be overcome by the addition of purified Factor V to the plasma of affected individuals (Dahlback and Hildebrand. 1994. Proc. Natl. Acad. Sci. USA. vol. 91. 1396-1400). Although this observation suggested linkage of APC-resistance to Factor V, no satisfactory explanation was given for the occurrence of resistance to APC at the molecular level.
If the cause or causes for said resistance could be identified, this would lead to a better understanding of thrombolytic disorders, as well as to better detection methods for such disorders and possibly to new and better ways of treatment or prophylaxis of said disorders and other disorders in the blood coagulation cascade.